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Description

Highlights

Cleaner System - sample degradation eliminated

Easy-to-use - protein expression complete in approximately two hours

Simple Analysis - protein can often be visualized directly on a Coomassie stained gel

A rapid method for gene expression analysis, PURExpress® is a novel cell-free transcription/translation system reconstituted from the purified components necessary for E. coli translation. With minimal nuclease and protease activity, the PURExpress system preserves the integrity of DNA and RNA templates/complexes and results in proteins that are free of modification and degradation. Transcription and translation are carried out in a one-step reaction, and require the mixing of only two tubes. With results available in a few hours, PURExpress saves valuable laboratory time and is ideal for high throughput technologies.

Figure 1: Protein expression using the PURExpress® In Vitro Protein Synthesis Kit
25 μl reactions containing 250 ng template DNA and 20 units RNase Inhibitor were incubated at 37°C for 2 hours. 2.5 μl of each reaction was analyzed by SDS-PAGE using a 10–20% Tris-glycine gel. The red dot indicates the protein of interest. Marker M is the Protein Ladder (NEB #P7703 ).
Figure 2: Incorporation of 35S-methionine enables visualizationof protein by autoradiography
25 μl reactions containing 250 ng template DNA, 20 units RNase Inhibitor and 2 μl 35S-met were incubated at 37°C for 2 hours. 2.5 μl of each reaction was analyzed by SDS-PAGE, the gel was fixed for 10 minutes, dried for 2 hours at 80°C and exposed to x-ray film for 5 hours at -80°C.
Figure 3: Schematic diagram of protein synthesis and purification by PURExpressFigure 4: Expression and reverse purification of DHFR (A) and T4 DNA Ligase (B) using PURExpress
125 μl reactions were carried out according to recommendations in the accompanying manual. Samples were analyzed on a 10–20% Tris-glycine gel and stained with Coomassie Blue. Note that in both cases, the desired protein can be visualized in the total protein fraction. The red dot indicates the protein of interest. Marker M is the Protein Ladder (NEB #P7703 ).

Kit Components

PURExpress Solution A

Control (DHFR) template (10 μl)

Solution B (75 μl)

Kit Components

The following reagents are supplied with this product:

Store at (°C)

Concentration

PURExpress Solution A

Control (DHFR) template

PURExpress Solution B

3.3X

Advantages and Features

Applications

Quickly generate analytical amounts of protein for further characterization

Properties and Usage

Storage Temperature

-80°C

Licenses

PURExpress® is based on the PURE System Technology originally developed by Dr. Takuya Ueda at the University of Tokyo and commercialized as the PURESYSTEM® by BioComber (Tokyo, Japan).

Licensed from BioComber (Tokyo, Japan) under Patent Nos. 7,118,883; WO2005-105994 and JP2006-340694. For research use only. Commercial use of PURExpress® In vitro Protein Synthesis Kit requires a license from New England Biolabs, Inc. This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.
This product is covered by one or more patents, trademarks and/or copyrights owned or controlled by New England Biolabs, Inc (NEB).

While NEB develops and validates its products for various applications, the use of this product may require the buyer to obtain additional third party intellectual property rights for certain applications.

For more information about commercial rights, please contact NEB's Global Business Development team at gbd@neb.com.

This product is intended for research purposes only. This product is not intended to be used for therapeutic or diagnostic purposes in humans or animals.

Supporting Documents

The following is a list of Safety Data Sheet (SDS) that apply to this product to help you use it safely.

The Product Manual includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name these document files: manual[Catalog Number].

The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at info@neb.com or fill out the Technical Support Form for appropriate document.

Notes

The DHFR control template is now supplied at 125 ng/µl. Use 2 µl for the positive control reaction. Template DNA, particularly plasmid DNA prepared by mini-prep (e.g. Qiagen) is often the major source of RNase contamination. We strongly recommend adding 20 units Murine RNase Inhibitor (NEB #M0314) to each reaction.

Add Solution B to Solution A, do not dilute Solution B unbuffered. We recommend a starting amount of 250 ng template DNA per 25 μl reaction. The optimal amount of input DNA can be determined by setting up multiple reactions and titrating the amount of template DNA added to the reaction. Typically, the optimal amount will fall in a range of 25–1000 ng template per 25 μl reaction.

Companion Products

Thaw and assemble reactions on ice
Thoroughly mix solutions A and B before using. Do not vortex Solution B or ribosomes, mix gently.
Solution A may have a cloudy white appearance. Add to the reaction as a uniform suspension.
Assemble the reactions in the following order on ice: Solution A, Solution B, RNAse Inhibitor, Water, Template DNA or RNA
Once reaction is assembled take time to make sure everything is thoroughly mixed by gently pipetting up and down, pulse spin and place at 37C for 2 to 4 hours.

The supporting documents available for this product can be downloaded below.